The functional improvement of plants including grains such as rice, has seen a significant development together with genetic engineering technology for plants. At first, improvements or plant breeding were directed to culture mainly targeting farmers, such as resistance against diseases, insects, herbicide and the like. Recently, targets have been changed to be directed to transformation of edible portions which can mainly appeal to consumers. Studies of expression in a plant of a variety foreign functional proteins, such as physiologically functional peptides and antibodies, have been performed worldwide, and in particular, seeds are of note as a producing machine of such a foreign functional protein, as seeds can preserve the protein for a long period of time in a stable manner (Japanese Laid-Open Publication No. 2002-58492; Molecular Breeding 9:149-158 (2002)). Further, studies for expression in seeds by the use of promoters have been reported (Plant Cell Physiol. 39:885-889 (1998)).
In grain seeds such as rice, the protein content thereof is said to be about several to about 10 percent, and most of the proteins are present as a storage protein. Storage proteins are believed to be a source for nitrogen nutrient in germination, but no physiological functions are believed to exist therein. Generally, proteins are classified into four categories depending on the solubility thereof, including glutelin, prolamin, globulin, and albumin. Types of storage proteins are unique for each protein species.
In seeds of Leguminosae plants, globulin is the major storage protein, whereas monocotyledonous seeds such as cereals, prolamin is the major storage protein with some exceptions (J. Exp. Botany 53:947-958 (2002)).
Rice exceptionally contains glutelin as a main storage protein amongst cereals, and the content thereof is 60-70% of the seed protein. The glutelin gene group consists of about ten genes per haploid genome, and these genes are classified into two subfamilies GluA and GluB, which have 60-65% homology at the amino acid level in the coding regions to each other. Further, each subfamily contains about five genes presenting 80% or greater homology in the amino acid level. Glutelin has evolutionary relationships with globulin of beans, a storage protein, and the amino acid composition thereof is relatively rich in essential amino acids and has high nutritional value. On the other hand, prolamin shares 20-30% of rice seed proteins, and thus the content thereof follows that of glutelin. Prolamin is characterized by a large amount of glutamin, and a relatively high content of proline. Prolamin is poor in lysine, an essential amino acid, and is poor in nutritive value. There are multiple genes for prolamins in the genome, with similar structures with slightly altered amino acid sequences, and it is estimated that the total number thereof is between 25 and 100. Another storage protein includes globulin having a molecular weight of 26 kDa, which shares sequence homologies at a few percent level of the storage proteins. In seeds, storage proteins are intracellularly accumulated in a granular cellular structure, called a protein body. If seeds are likened to a factory of protein production, the protein body plays a role as a protein warehouse. Rice is characterized in that two types of protein bodies co-exist in albumen, which have completely different origins and purpose, and are called, Protein Body 1 (in which prolamin is accumulated), and Protein Body 2 (in which glutelin and globulin are accumulated), respectively (Plant Cell Physiol. 28: 1517-1527 (1987); Plant Physiol. 88: 649-655 (1988); Plant Biotechnology, 16: 103-113 (1999); Jokyo (1993) 414-420; Inegakutaisei Vol 3., 32-39 (1990), and Inegakutaisei Vol. 3 317-325 (1990)).
An attempt to modify a storage protein has been made for a variety of cereals, and these attempts were mainly directed to improve nutritional value or processing characteristics. In the case of rice, low-protein content is preferred as raw material for sake production or rice processed foods, and extensive search has been conducted to find variant lineages with reduced main seed storage protein content or composition amongst rice variant pools which were subjected to radiation or mutagen treatment (Iida, S. et al. (1997) Theor. Appl. Genet. 94, 177-183; Iida, S. et al. (1993) Theor. Appl. Genet. 87, 374-378; Crop Sci. 39: 825-831 (1999); Ikushugaku Kenkyu 4 (Suppl. 1) 66 (2002); Ikushugaku zasshi 47 (Suppl. 2) 632 (1997); Ikushugaku Zasshi 47 (Suppl. 2) 176 (1997); Ikushugaku Zasshi 45 (Suppl. 2) 502 (1995); and Ikushugaku Zasshi 45 (Suppl. 1) 508 (1995)). To date, variant rice which lack specific glutelin gene or which shows reduced glutelin content have been subjected to extensive research, due to the fact that glutelin is the major storage protein of rice and the major target thereof. Typical low glutelin rice lineages include mutant LGC-1 and recombinant glutelin rice lineages by antisense technology as known in Japanese Patent No. 3149951; Ikushugaku Kenkyu 3 (2001) and Molecular Breeding 8: 273-284 (2001)). Amongst these, LGC-1 has been analyzed to prove that the causative gene of variation is dominantly inherited by one gene, and the gene has been recently isolated (The Plant Cell 15, 1455-1467, (2003)). The structure thereof lacks both terminator regions between two glutelin genes (GluB4 and GluB5) in the genome regions, which originally are to be transcribed to each other, thereby the transcription of GluB4 promoter was transcribed in an antisense direction beyond its original termination point to the GluB5 gene, resulting in double-stranded RNA formation of homologous regions of GluB4 and GluB5, and expression suppression of the entire storage protein glutelins by RNAi phenomenon. Mutations per se may be used for crossing or breeding species, and the variety per se or its progeny are registered as a novel plant variety (Nogyogijutsu 55 (10), 26-29 (2000) and Ikushugakukenkyu 4: 33-42 (2002)). The LGC-1 gene, however, is not recognized to have an optimum structure for suppressing glutelin expression, and thus LGC-1 still retains glutalin at about 30-50% of its original content. The present invention is significantly superior, in terms of technology, in that the RNAi phenomenon is used for suppressing expression of a storage protein by means of an optimum structure gene construct, whereby significant improvement in efficiency of expression inhibition has been achieved. A common problem shared by low-glutelin rice lineages is that the glutelin content is significantly reduced compared to the original content, but a significant increase in prolamins has been observed. This is due to the regulatory machinery in plants to maintain protein content in the seed at a certain level, and a plant senses that the lack of glutelin to induce an increase in the prolamin synthesis. In this sense, low-glutelin lineages have not been fully successful in protein reduction, even with a variant rice in which storage protein composition has been altered.
On the other hand, prolamins are low in nutrition, have poor digestibility, and lower working characteristics or taste of rice, and thus are desired to be lowered. Although some rice variants are already known in the art (Iida, S. et al. (1997) Theor. Appl. Genet. 94, 177-183; Iida, S. et al. (1993) Theor. Appl. Genet. 87, 374-378; Ikushugaku kenkyu (Supple. 1) 66 (2002); Ikushugaku zasshi 47 (Suppl. 2) 632 (1997); Ikushugaku zasshi 47 (Supple. 2) 176 (1997); Ikushugaku zasshi 45 (Supple. 2) 502 (1995); and Ikushugaku zasshi 45 (Supple. 1) 508 (1995) 15˜19), the degree of reduction in prolamins is small, or other variation had also occurred and thus the plants became infertile, and therefore selection of promising lineages has not yet been done. Therefore, there cannot be any clue to date to solve problems where prolamins are significantly increased which is accompanied by the reduction of a group of glutelin lineages. As such, development of low prolamin rice is a problem that has yet to be solved.
As mentioned in the introduction, research has focused using seed production machinery (bioreactor) for production of useful proteins. Seeds have been used throughout history as a food eaten by human beings. There is little risk in which toxic substances contaminate seeds, and also such seeds have merits in that they can be eaten without specific purification or operation. If seeds with reduced storage proteins are used, remaining amino acids are efficiently used for synthesizing foreign protein synthesis, and thus there is a possibility in which expression thereof increases. Even if storage proteins are reduced, and the same amount are replaced with useful proteins, function as a nitrogen source is fulfilled and thus it is believed that there is no problem in terms of seed physiology. It is therefore, contemplated that as, glutelin and prolamin are accumulated in separate granules in a strict manner in rice, and such machinery can be successfully used to increase expression of a useful protein by accumulating the protein into the protein body, or facilitating purification of a useful protein. For example, a signal sequence of the storage protein prolamin has high homology not only within the same species, but also between species, for example, wheat, barley or maize storage proteins, and thus these sequences may be used to accumulate proteins in Protein Body 1.
However, research to date in which a foreign protein is expressed in seeds for creating a functional crop (Japanese Laid-Open Publication No.: 2002-17187; Japanese Laid-Open Publication No.: 7-213185; and Japanese PCT National Phase Laid-Open Publication No.: 2001-518305; Ikushugaku kenkyu 5 (Suppln. 2), 294 (2003)), usually uses normal varieties for expression thereof. In such cases, most of amino acid pool in the seeds is consumed for rice storage protein synthesis, and thus the amount that can be used for useful protein production is very limited. As a result, useful protein expression level is limited and thus efficiency of functional modification is not good. Further, in the example where storage protein variant rice is used for foreign protein expression (Japanese Laid-Open Publication No.: 2002-58492), the total amount of seed protein of glutelin-reduced rice is equal to that in the original, and thus the problem of competition between useful protein and storage protein in the seeds is not solved. As such, conventional technology is not recognized to use an efficient expression system for a foreign protein.
Low-protein rice is useful when rice is used for removing proteins thereof in any manner since it reduces labor-intensity for removing proteins. In general, rice as food or a raw material for rice-based processed food, low-protein rice would be preferable as a raw material for allergen-reduced food, which has become increasingly required in recent years (“Bioscience of seeds”, Shushi Seirikagaku kenkyukai (Seed physiochemical research association) ed. Gakkai center 2., Rice, 251-257 (1995); (“Bioscience of seeds”, Shushi Seirikagaku kenkyukai (Seed physiochemical research association) ed. Gakkai center 4, Processed food product of rice 359-365 (1995), and (“Bioscience of seeds”, Shushi Seirikagaku kenkyukai (Seed physiochemical research association) ed. Gakkai center 5, Sake, 366-371, (1995)). Some one third of Japanese people have an allergy, and amongst these people, rice allergies, for which few problems had been reported to date, are being increasingly reported. In such cases, nutritional replacement food substitutes are required; however, physiological problem is significant as conventionally consumed rice cannot be eaten. As such, there is an increasing need and demand for processed rice based on allergen reduced for rice allergy patients. Globulin protein is the major allergen in rice. Japanese Laid-Open Publication No. 2-167040 attempts degradation and removal of globulin protein by reacting rice with proteinase in order to remove allergen removal. Further, Japanese Patent No. 3055729 describes a method for solubilizing and removing allergen by alkaline washing “low-protein rice” as a raw material. In either case, protein removal is the major object, and the protein extract and removal efficiency would be higher if rice with lower protein content is used. However, rice lineages actually used are normal lineages, or those in which the storage protein composition has been altered but the protein amount per se is not reduced, and thus it is not efficient. Japanese patent No. 3055729 attempts a variety of known variant rice, and does not attempt to develop functional crops in a strategic manner. Further, low-glutelin or low-prolamin contents, which have a large range of molecular weights in a single variety, have not been established in a single variety, which is considered to be preferable, and thus the removal of all allergens has not been successful to date.
As described above, there is high demand for providing a low-protein content seed, and improving foreign protein expression in a seed in the field of high utility rice and rice processed foods, production of functional plant and seed and the like.
It is thereof an object of the present invention to provide a method for reduce protein content in seeds and development of necessary technology therefore, and thus developed plants and seeds thereof, and method of using such plants and seeds.